PI: Arlen W Johnson Summary Ribosomes are responsible for the rapid and accurate production of all proteins in cells in all forms of life on earth. The ability of these molecular machines to carry out faithful translation depends on their complex structure that allows dynamic interaction with ligands. The mature ribosome in eukaryotic cells is composed of two parts, the large 60S subunit that carries out polypeptide synthesis and the small 40S subunit that decodes mRNA. The assembly of a eukaryotic ribosome involves over 200 accessory assembly factors, whose function, in many cases, is still unknown. Considering the complexity of ribosome structure and function and its critical role in decoding our genetic information, ensuring their correct assembly would seem a necessary but daunting task for cells. Lately, considerable interest has been focused on mechanisms of quality control in the ribosome biogenesis pathway. This proposal focuses on two distinct topics within ribosome assembly; (1) completion of the peptidyl transferase center of the 60S subunit and the mechanisms for assessing its functional integrity and (2) the transition from the early 90S pre-ribosomal precursor to the pre-40S precursor. This proposal is directed at understanding the quality control mechanisms that assess the functional and structural integrity of the peptidyl transferase center of the subunit, upon insertion of ribosomal protein Rpl10. This proposal builds on our recent determination of the atomic structure of a preribosome and release of two factors, Tif6 and Nmd3. The release of Nmd3 and Tif6 is dependent on the two GTPases Efl1 and Lsg1 and constitutes the primary quality control check point in during 60S maturation. In humans, defects in the quality control step lead to T-cell acute lymphoblastic leukemia and Shwachman-Diamond syndrome. Assembly of the small ribosomal subunit involves stepwise cotranscriptional assembly of the 90S particle, a large protein-RNA complex, scaffolded on U3-snoRNA. However, the presence of U3 is mutually incompatible with the final folded structure of small subunit RNA and must be removed once transcription of the RNA is complete and the 90S particle has fully assembled. The transition from the 90S to pre-40S is poorly understood. We propose that the displacement of U3 by the RNA helicase Dhr1 is a primary event that drives the transition of the 90S into the pre-40S particle. We will determine how the activity of Dhr1 is regulated to ensure the timely release of U3.